1. Muscle Cells Provide Instructions for Planarian Regeneration
Jessica N. Witchley, Mirjam Mayer, Daniel E. Wagner, Jared H. Owen, Peter W. Reddien 
Cell Reports 
Volume 4, Issue 4, Pages (August 2013)
DOI: /j.celrep
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2. Cell Reports 2013 4, 633-641DOI: (10.1016/j.celrep.2013.07.022)
Copyright © 2013 The Authors Terms and Conditions

3. Figure 1 Positional Conflict without Neoblasts
(A) Anterior cylindrical plugs from animals irradiated with 6,000 rad were transplanted into the posterior of unirradiated hosts. For other transplant experiments, see Kato et al., 2001; Saló and Baguñà, 1985.
(B) Tissue outgrowth was triggered by transplantation (yellow asterisk).
(C) ndl-3 and wntP-2 were respectively expressed in the anterior and posterior of wild-type animals, and in the distal and proximal regions of an outgrowth.
(D) Cartoon depicting domains of PCG expression, which define positions across AP, DV, and ML body axes (modified from Reddien, 2011).
See also Figure S1.
Cell Reports 2013 4, DOI: ( /j.celrep )
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4. Figure 2 PCGs Are Coexpressed in Subepidermal Cells
(A) Expression patterns of the PCGs analyzed in this figure (see also Figure S1). Cartoon depicts seven analyzed zones (zones 1–7). Anterior: up; left animal: dorsal view; right animal: ventral view. Animals are 1.5–2.5 mm in length. e, eye; m, mouth; px, pharynx.
(B) Fluorescent images of transverse tissue sections. Dorsal up, as illustrated. ep, epidermis.
(C) Three RNA probe pairs were used in double FISH experiments. The percentage of cells expressing the less-abundant (fewer cells) gene that also expressed the more-abundant gene is shown (the less-abundant gene is green in each pair). Three animals were counted and the data were summed for each gene pair. Nuclear signal (DAPI) is shown in blue.
Scale bar, 10 μm (B and C). See also Figures S2 and S3.
Cell Reports 2013 4, DOI: ( /j.celrep )
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5. Figure 3 PCGs Are Expressed in Muscle Cells
(A) Prepharyngeal sections (dorsal up; compare with the cartoon) show that position control cells (sFRP-2+ or nlg-8+) are distinct from specific peripheral neoblast progeny cell types (i, NB.21.11e+; ii, agat-1+), peripheral neurons (iii, chat+), intestinal cells (iv and v, mat+), and protonephridia (vi, rootletin+). Internal sFRP-2+ cells close to intestine (iv) and nlg-8+ cells far from intestine (v) are shown. ep, epidermis.
(B) collagen is broadly expressed subepidermally, similar to the density and location of position control cells (dorsal view shown; anterior up).
(C) collagen+ cells are muscle cells that coexpress troponin both subepidermally (insets 1 and 3) and internally (inset 2).
(D) collagen+ cells coexpress both tropomyosin and troponin. The percentage of collagen+ cells (>500 cells counted from three animals each) expressing troponin/tropomyosin is shown. troponin and tropomyosin are also coexpressed in the same cells. Right: cross-section showing subepidermal troponin+ cells with signal extending into the region of muscle fibers (labeled with the anti-myosin-heavy-chain (MHC) antibody TMUS-13; Cebrià and Vispo, 1997).
(E) PCGs are coexpressed with troponin (the percentage of position control cells expressing troponin is shown; >250 position control cells were examined from three animals each). Axis regeneration genes notum (anterior regeneration), wnt1 (posterior regeneration), and bmp4 (dorsal regeneration) were also coexpressed with troponin (115, 41, and 327 cells, respectively, were examined). Additional examples are provided in Figure S3B.
(F) Differential interference contrast (DIC, top) and fluorescent micrograph (bottom) of isolated muscle fibers expressing troponin and PCGs (six sFRP-2+, four nlg8+, and four wntP-2+ cells were examined). Nuclei (DAPI) are blue. Additional green signal is in debris (lacking a nucleus).
(G) Coexpression of 19 PCGs (red, combined RNA probes) and collagen (green) in zones 1–7 (as in Figure 2). The percentage of double-positive cells is given as a fraction of all PCG-positive cells (red) and all collagen+ cells (green; three animals examined for each region). Neural netrin1 and netrin2 expression affects the percentage of red cells that are collagen+ in zones 6 and 7.
Scale bars, 10 μm (A, C [bottom], and D–F), 100 μm (B and C [top]), 200 μm (E), and 20 μm (G). See also Figure S1.
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6. Figure 4 Dynamic PCG Expression in Muscle Cells following Amputation
(A) Animals were irradiated with 6,000 rads (eliminating neoblasts) and amputated transversely 3 days later. The percentage of PCG+ cells at wounds expressing collagen or troponin was examined in three animals each. Anterior-facing wounds are from tails, and posterior-facing wounds are from heads (except for nlg-1 and glypican, where one anterior-facing wound was from a trunk; 117–547 cells were examined for each condition); 108/108 and 423/425 collagen+ cells were troponin+ at anterior- and posterior-facing wounds, respectively, at 6 hours postamputation (6 hpa; 168/168 and 141/141 were double positive at 16 hpa).
(B) Schematic illustrating the surgical procedure. Tail fragments were fixed at 16 hpa, labeled by FISH and DAPI, and analyzed in sagittal sections. Anterior is left. Bottom: zoomed images from the anterior-facing wound site. Arrowheads indicate coexpression of notum and collagen in cells adjacent to the amputation plane (n = 418/425 were double positive).
(C) PCGs with regeneration RNAi phenotypes are coexpressed with collagen or troponin in blastemas (the time after amputation is indicated).
(D) Transient coexpression of anterior-specific (wnt2) and posterior-specific (wntP-2) PCGs during regeneration. Animals were irradiated (6,000 rads) and amputated 3 days later. Left: intact animal (3 days postirradiation). Center: tail fragments (bottom, zoomed) at 0 dpa and 4 dpa. wnt2 was coexpressed with wntP-2 at 4 dpa. Right: Triple-color FISH demonstrates coexpression of wnt2, wntP-2, and collagen.
(E) wnt1(RNAi) animals had clusters of numerous ovo+ cells at posterior-facing wounds at 4 dpa (n = 2/5 animals, with three total clusters present and none present in control RNAi [n = 4] animals; see also Figure S4E). All posterior ovo+ cell clusters in wnt1(RNAi) animals possessed multiple smedwi-1+/ovo+ cells (arrowheads; smedwi-1 expression marks neoblasts).
(F) Model. PCG expression in muscle specifies the identity of new cell types made in tissue turnover. Following amputation, muscle cells change their PCG expression, and these changes dictate which type of new tissue is regenerated. The influence of muscle on neoblasts is depicted by purple and green signaling environments, but it need not be direct.
Scale bars, 20 μm (A), 50 μm (B), and 10 μm (C and D). See also Figure S1.
Cell Reports 2013 4, DOI: ( /j.celrep )
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7. Figure S1
Expression Patterns of the PCG Genes Used in this Study, Related to Figures 1, 2, 3, and 4
(A) In situ hybridizations on wild-type animals with PCG genes and other anatomical markers used in this manuscript. For each gene, two images are shown. Left, dorsal view; right, ventral view. Anterior is to the top in all images; all animals are mm in length. The color of the labeling indicates which signaling pathway the gene is part of. In addition to PCGs, the figure also depicts the expression pattern of other tissue markers used in this manuscript, namely smedwi-1 (Reddien et al., 2005b), agat-1 (Eisenhoffer et al., 2008), chat (Wagner et al., 2011), mat (Wenemoser and Reddien, 2010), and mhc-1. rootletin (Glazer et al., 2010) was used to mark nephridia in Figure 3.
(B) Position control genes are expressed in a subepidermal layer, peripheral to neoblasts. Top row: prepharyngeal sections showing the subepidermal localization of GPS cells marked by the combined expression of 10 position control genes (PCGs; the same as used in Figure 2A) in red and the more interiorly localized neoblasts marked by the expression of smedwi-1 in green. Bottom two rows: Sections showing the expression of smedwi-1 in green and individual position control genes in red in the respective regions of their expression. Note that in most cases shown here, the epidermis is missing (due to the experimental procedure).
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8. Figure S2 PCGs Are Not Expressed in Neoblasts, Related to Figure 2
(A) Position control gene expression is not irradiation sensitive. For each gene, two images are shown. Left, dorsal view; right, ventral view. Anterior is to the top in all images; all animals are mm in length. Animals were irradiated with 6,000 rad and fixed three days later. Animals were then labeled with all markers used in this manuscript. smedwi-1 is expressed in neoblasts, and lack of smedwi-1 + cells demonstrates irradiated eliminated neoblasts.
(B) Effects of irradiation on PCG expression levels. Levels of PCG gene expression in whole animal tissues 12, 24, 36, and 48 hr post-irradiation (6,000 Rads) were obtained from microarray data sets available in GEO, accession number GSE34969 (Wagner et al., 2012). Expression levels are represented as log2 values of mean intensity ratios (irradiated/unirradiated). Left, Shown are control gene expression levels for differentiated tissues (Smed-chat and Smed-mat), which remain unchanged following irradiation. Also shown are representative markers of neoblasts (Smed-bruli, Smed-pcna, smedwi-1, Smed-mcm2) and neoblast progeny cell types (NB.32.1G, NB,21.11E), which are rapidly depleted within 48 hr of irradiation. Right, Expression profiles of PCGs following irradiation. Control genes use same color scheme as at left.
Cell Reports 2013 4, DOI: ( /j.celrep )
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9. Figure S3
Analysis of PCG Expression, with Controls for FISH, Related to Figures 2 and 3
(A) wnt2/netrin1, netrin2/nlg-7, nlg-7/slit, netrin2/nlg-7, and admp/slit displayed overlapping expression in the same cells. Overlap at lower levels was observed for netrin1/sFRP-2, netrin1/nlg-7, and little overlap was observed between netrin1/netrin2. Therefore, a subpopulation of position control cells expresses netrin2/sFRP-2/nlg-7 at high frequencies, and another coexpresses netrin1/wnt2 at high frequencies, with some overlap between these two populations. Scale bar, 10 μm.
(B) Additional examples of position control genes expressed in muscle. Position control genes are coexpressed with troponin. The zone in which the image was taken is indicated (compare schematic in Figure 2). For wnt5 expression, lateral expression in a transverse tissue section is shown. Scale bar, 10 μm.
(C–E) Controls for fluorescent in situ hybridizations. Shown are representative confocal projections from whole-mount fluorescent in situ hybridization (FISH) experiments. DIG probes were developed first using anti-DIG-POD and rhodamine tyramide (magenta). Peroxidase activity was then quenched using either 1% sodium azide or 4% formaldehyde in PBSTx (see methods). Fluorescein probes were developed second using anti-Fluor-POD and FITC tyramide (green), and DAPI stained (blue).
(C) Representative dorsal projection (anterior up) showing bmp4 expressing cells expressing collagen. Scale bars, 20 μm. Asterisks indicate pigment cups. Representative ventral projection, anterior left.
(D) Coexpression of position control genes (PCGs) ndk, ndl-3, ndl-4, notum, sFRP2, wnt2, wntA, wnt11-1, wnt11-2, wntP-2, wntP-1, wnt5, bmp4, nlg7, slit, admp (magenta) with collagen (green) is shown. Scale bars 200 μm (left), 20 μm (insets).
(E) Representative confocal projections from animals labeled for PCG expression as in (D) using the 4% formaldehyde inactivation method. The extent of green fluorescence due to residual HRP activity following inactivation is shown in the right column of images (collagen probe omitted). Left and right image panels were collected with identical pinhole, laser, and gain settings on a Zeiss LSM 700 scanning confocal microscope.
(F–J) Positional control gene expression outside the subepidermal layer. Scale bar, 10 μm.
(F) Tissue sections (see schematic inset) display sFRP-2 and nlg-8 expression around the intestine, and these cells also express troponin/collagen. Percentage of position control gene+ cells expressing troponin/collagen is shown (>500 total position control gene+ cells examined from three animals each).
(G) sFRP-1 and ndl-4 are expressed within the pharynx in pharyngeal muscle cells (troponin+).
(H) wntP-2 is expressed strongly internally at the anterior end of the pharynx in cells that also express troponin.
(I) wnt1 and wntA display brain region expression that is not in muscle cells.
(J) bmp4 is expressed in a small ring of troponin- cells at the anterior end of the pharynx.
(G–J) Anterior, up.
Cell Reports 2013 4, DOI: ( /j.celrep )
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10. Figure S4
Expression and Role of PCGs in Regeneration, Related to Figure 4
(A) Animals (not irradiated) were examined at 6 or 16 hr after amputation as indicated, depending upon the peak time of wound-induced expression (21). Wound-induced genes were coexpressed together with troponin.
(B) bmp4 expression at 24hpa in parasagittal fragments, as indicated in the schematic. Percentage overlap quantified from three unirradiated parasagittal animal fragments.
(C) Analysis of PCG and collagen coexpression in sagittal sections. Schematic illustrates surgical procedure. Transverse amputations were performed to produce fragments for analysis of wound-induced PCG expression: a tail fragment containing an anterior-facing wound site (see Figure 4), and a “pre-pharyngeal” (prePHX) fragment containing both anterior and posterior-facing wound sites. In tail fragments, 95.7% of wnt1-expressing cells also expressed collagen (n = 180/188) and 98.4% of notum-expressing cells also expressed collagen (n = 418/425). Tissue fragments were fixed 16 hr post-amputation, labeled by FISH and DAPI, and analyzed in sagittal sections. Zoomed images from 16 hpa pre-pharyngeal and tail fragments assessing PCG and collagen coexpression in multiple wound locations and orientations. Asterisks indicate approximate amputation site. Dotted lines indicate animal boundary. Scale bars, 50 μm.
(D) The distribution of notum+ / collagen+ and wnt1+ / collagen+ cells along the AP axis of tail fragments is shown in 25 μm bins.
(E–G) Defects in neoblast eye lineage-specification following notum or wnt1 RNAi. ovo is expressed in eye progenitors during eye head regeneration, including in neoblasts. During regeneration, wnt1 and notum were expressed in muscle cells at wounds and were required for formation of normal ovo+ neoblast numbers at wounds (normally none at posterior and numerous at anterior-facing wounds).
(E) 2/5 wnt1(RNAi) animals had ovo+ clusters of cells at posterior-facing wounds 4 days following amputation (3 total clusters were present, and none were present in control RNAi (n = 4) animals). All three posterior ovo+ cell clusters possessed multiple smedwi-1+ / ovo+ cells (see Figure 4E). Anterior, left.
(F) notum(RNAi) animals had too few notum+ cells at anterior-facing wounds 48 hr following amputation (n ≥ 7 anterior blastema left or right sides were counted; shown are means ± SDs; p < 0.005, unpaired t test). Anterior, left.
(G) Optical sections of anterior-facing wounds show example smedwi-1+ / ovo+ cells (arrowheads). From sections spanning the entirety of the eyes shown (only sections are shown) 21/50 ovo+ cells were also smedwi-1+ in the control and 4/11 ovo+ cells were also smedwi-1+ in notum RNAi. Anterior, left for the control and down for notum RNAi. Bars, 20 μm.
Cell Reports 2013 4, DOI: ( /j.celrep )
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